Afinitor, Everolimus

Afinitor (everolimus)

40-O-(2-hydroxyethyl)-rapamycin

Company: Novartis
Approval Status: Approved July 2012
Treatment Area: hormone receptor-positive, HER2-negative breast cancer

Everolimus is a derivative of Rapamycin (sirolimus), and works similarly to Rapamycin as an mTOR (mammalian target of rapamycin) inhibitor. It is currently used as an immunosuppressant to prevent rejection of organ transplants. In a similar fashion to other mTOR inhibitors Everolimus' effect is solely on the mTORC1 protein and not on the mTORC2 protein.

Also known as: Afinitor, Certican, Zortress, SDZ-RAD, RAD001, Everolimus [USAN], 42-O-(2-Hydroxyethyl)rapamycin, RAD 001

Molecular Formula: C₅₃H₈₃NO₁₄   Molecular Weight: 958.22442

159351-69-6  CAS NO

BRANDS

Afinitor	Novartis
Certican	Novartis
VOTUBIA	Novartis
Zortress	Novartis

Afinitor (everolimus), an inhibitor of mTOR (mammalian target of rapamycin), is an antineoplastic agent.

Afinitor is specifically approved for the treatment of postmenopausal women with advanced hormone receptor-positive, HER2-negative breast cancer (advanced HR+ BC) in combination with exemestane, after failure of treatment with letrozole or anastrozole.

Afinitor is supplied as a tablet for oral administration. The recommended dose of Afinitor for breast cancer is 10 mg, to be taken once daily, at the same time every day, either consistently with food or consistently without food.

FDA Approval
The FDA approval of Afinitor for the treatment of advanced hormone receptor-positive, HER2-negative breast cancer was based on a randomized, double-blind, multicenter study in 724 postmenopausal women with estrogen receptor-positive, HER 2/neu-negative advanced breast cancer with recurrence or progression following prior therapy with letrozole or anastrozole.

Everolimus is indicated for the treatment of postmenopausal women with advanced hormone receptor-positive, HER2-negative breast cancer (advanced HR+ BC) in combination with exemestane, after failure of treatment with letrozole or anastrozole. Indicated for the treatment of adult patients with progressive neuroendocrine tumors of pancreatic origin (PNET) with unresectable, locally advanced or metastatic disease. Indicated for the treatment of adult patients with advanced renal cell carcinoma (RCC) after failure of treatment with sunitinib or sorafenib. Indicated for the treatment of adult patients with renal angiomyolipoma and tuberous sclerosis complex (TSC), not requiring immediate surgery. Indicated in pediatric and adult patients with tuberous sclerosis complex (TSC) for the treatment of subependymal giant cell astrocytoma (SEGA) that requires therapeutic intervention but cannot be curatively resected.

Everolimus (RAD-001) is the 40-O-(2-hydroxyethyl) derivative of sirolimus and works similarly to sirolimus as an inhibitor of mammalian target of rapamycin (mTOR).

It is currently used as an immunosuppressant to prevent rejection of organ transplants and treatment of renal cell cancer and other tumours. Much research has also been conducted on everolimus and other mTOR inhibitors for use in a number of cancers.

It is marketed by Novartis under the tradenames Zortress (USA) and Certican (Europe and other countries) in transplantation medicine, and Afinitor in oncology.

EVEROLIMUS

AFINITOR (everolimus), an inhibitor of mTOR, is an antineoplastic agent.

The chemical name of everolimus is (1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18- dihydroxy-12-{(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl}-19,30-dimethoxy15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-aza-tricyclo[30.3.1.0^4,9]hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20pentaone.

The molecular formula is C₅₃H₈₃NO₁₄ and the molecular weight is 958.2. The structural formula is:

AFINITOR Tablets are supplied for oral administration and contain 2.5 mg, 5 mg, 7.5 mg, or 10 mg of everolimus. The tablets also contain anhydrous lactose, butylated hydroxytoluene, crospovidone, hypromellose, lactose monohydrate, and magnesium stearate as inactive ingredients.

AFINITOR DISPERZ (everolimus tablets for oral suspension) is supplied for oral administration and contains 2 mg, 3 mg, or 5 mg of everolimus. The tablets for oral suspension also contain butylated hydroxytoluene, colloidal silicon dioxide, crospovidone, hypromellose, lactose monohydrate, magnesium stearate, mannitol, and microcrystalline cellulose as inactive ingredients.

1. R.N Formica Jra, K.M Lorberb, A.L Friedmanb, M.J Biaa, F Lakkisa, J.D Smitha, M.I Lorber (March 2004). “The evolving experience using everolimus in clinical transplantation”. Elsevier 36 (2): S495–S499.
2.  “Afinitor approved in US as first treatment for patients with advanced kidney cancer after failure of either sunitinib or sorafenib” (Press release). Novartis. 2009-03-30. Retrieved April 6, 2009.
3. “Novartis receives US FDA approval for Zortress (everolimus) to prevent organ rejection in adult kidney transplant recipients” (Press release). Novartis. 2010-04-22. Retrieved April 26, 2010.
4. “Novartis’ Afinitor Cleared by FDA for Treating SEGA Tumors in Tuberous Sclerosis”. 1 Nov 2010.
5. http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm254350.htm
6. “US FDA approves Novartis drug Afinitor for breast cancer”. 20 Jul 2012.

PATENTS

Country	Patent Number	Approved	Expires (estimated)
United States	6440990	1993-09-24	2013-09-24
Canada	2145383	2004-11-16	2013-09-24
Canada	2225960	2004-05-11	2016-07-12
United States	7297703	1999-12-06	2019-12-06

US2011262525	10-28-2011	METHODS OF TREATMENT
US2011014117	1-21-2011	ANTI-IGF1R
US2011009387	1-14-2011	HISTONE H2AX (HH2AX) BIOMARKER FOR FTI SENSITIVITY
US7682647	3-24-2010	Thermal treatment of a drug eluting implantable medical device
US7645747	1-13-2010	Therapeutic phosphonate compounds
US7605257	10-21-2009	Processes for preparing water-soluble polyethylene glycol conjugates of macrolide immunosuppressants
US2009258054	10-16-2009	Heparin Prodrugs and Drug Delivery Stents Formed Therefrom
US2009227543	9-11-2009	PHOSPHONATE COMPOUNDS HAVING IMMUNO-MODULATORY ACTIVITY
US7470724	12-31-2008	Phosphonate compounds having immuno-modulatory activity
US7432261	10-8-2008	Anti-inflammatory phosphonate compounds

US2008152644	6-27-2008	Genes Involved in Neurodegenerative Conditions
US7285304	10-24-2007	Fluid treatment of a polymeric coating on an implantable medical device
US7241771	7-11-2007	Oxepane isomer of 42-O-(2-hydroxy)ethyl-rapamycin
US2007032853	2-9-2007	40-O-(2-hydroxy)ethyl-rapamycin coated stent
US2007004767	1-5-2007	Methods for treating neurofibromatosis 1
US2006199788	9-8-2006	Anti-inflammatory phosphonate compounds

WO1994009010A1	Sep 24, 1993	Apr 28, 1994	Sandoz Ag	O-alkylated rapamycin derivatives and their use, particularly as immunosuppressants
WO2007135397A1 *	May 18, 2007	Nov 29, 2007	Christoph Beckmann	36 -des (3 -methoxy-4 -hydroxycyclohexyl) 36 - (3 -hydroxycycloheptyl) derivatives of rapamycin for the treatment of cancer and other disorders
EP0663916A1	Sep 24, 1993	Jul 26, 1995	Novartis AG	O-alkylated rapamycin derivatives and their use, particularly as immunosuppressants
US5665772	Sep 24, 1993	Sep 9, 1997	Sandoz Ltd.	O-alkylated rapamycin derivatives and their use, particularly as immunosuppressants
US20030125800	Apr 24, 2002	Jul 3, 2003	Shulze John E.	Drug-delivery endovascular stent and method for treating restenosis

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Rapamycin is a known macrolide antibiotic produced by Streptomvces hvgroscopicus. having the structure depicted in Formula A:

See, e.g., McAlpine, J.B., et al., J. Antibiotics (1991) 44: 688; Schreiber, S.L., et al., J. Am. Chem. Soc. (1991) J_13: ⁷⁴³³'- US Patent No. 3 929 992. Rapamycin is an extremely potent immunosuppressant and has also been shown to have antitumor and antifungal activity. Its utility as a pharmaceutical, however, is restricted by its very low and variable bioavailabiiity as well as its high toxicity. Moreover, rapamycin is highly insoluble, making it difficult to formulate stable galenic compositions.

Everolimus, 40-O-(2-hydroxyethyl)-rapamycin of formula (1) is a synthetic derivative of rapamycin (sirolimus) of formula (2), which is produced by a certain bacteria strain and is also pharmaceutically active.

(1)                                                                                                               (2)

Everolimus is marketed under the brand name Certican for the prevention of rejection episodes following heart and kidney transplantation, and under the brand name Afinitor for treatment of advanced kidney cancer.

Due to its complicated macrolide chemical structure, everolimus is, similarly as the parent rapamycin, an extremely unstable compound. It is sensitive, in particular, towards oxidation, including aerial oxidation. It is also unstable at temperatures higher than 25°C and at alkaline pH.

Everolimus and a process of making it have been disclosed in WO 94/09010

Synthesis

Alkylation of rapamycin (I) with 2-(tert-butyldimethylsilyloxy)ethyl triflate (II) by means of 2,6-lutidine in hot toluene gives the silylated target compound (III), which is deprotected by means of 1N HCl in methanol (1). (Scheme 21042401a) Manufacturer Novartis AG (CH). References 1. Cottens, S., Sedrani, R. (Sandoz-Refindungen VmbH; Sandoz-Patent GmbH; Sandoz Ltd.). O-Alkylated rapamycin derivatives and their use, particularly as immunosuppressants. EP 663916, EP 867438, JP 96502266, US 5665772, WO 9409010.EP 0663916; EP 0867438; JP 1996502266; JP 1999240884; US 5665772; WO 9409010

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SYNTHESIS

https://www.google.com/patents/WO2012103960A1

(US 5,665,772, EP 663916). The process principle is shown in the scheme below, wherein the abbreviation RAP-OH has been used as an abbreviation for the rapamycin structure of formula (2) above, L is a leaving group and P is a trisubstituted silyl group serving as a OH- protective group.

RAP-OH + L-CH₂-CH₂-0-P — --> RAP-O-CH2-CH2-O-P — - > RAP-O-CH2-CH2-OH

(2)                                                 (4)                                                                 (1)

Specifically, the L- group is a trifluoromethanesulfonate (triflate) group and the protective group P- is typically a tert-butyldimethylsilyloxy- group. Accordingly, the known useful reagent within the above general formula (3) for making everolimus from rapamycin is 2-(tert-butyldimethylsilyloxy)ethyl triflate of formula (3 A):

According to a known synthetic procedure disclosed in Example 8 of WO 94/09010 and in Example 1 of US application 2003/0125800, rapamycin (2) reacts in hot toluene and in the presence of 2,6-lutidine with a molar excess of the compound (3 A), which is charged in several portions, to form the t-butyldimethylsilyl-protected everolimus (4A). This compound is isolated and deprotected by means of IN aqueous HC1 in methanol. Crude everolimus is then purified by column chromatography. Yields were not reported.

(2)                                       (3A)                              (4A)                                (1)

In an article of Moenius et al. (J. Labelled Cpd. Radiopharm. 43, 113-120 (2000)), which used the above process for making C14-labelled and tritiated everolimus, a diphenyl- tert.butylsilyloxy -protective group was used as the alkylation agent of formula (3B).

Only 8% yield of the corresponding compound (4B)

and 21% yield of the compound (1) have been reported.

Little is known about the compounds of the general formula (3) and methods of their preparation. The synthesis of the compound (3 A) was disclosed in Example 1 of US application 2003/0125800. It should be noted that specification of the reaction solvent in the key step B of this synthesis was omitted in the disclosure; however, the data about isolation of the product allow for estimation that such solvent is dichloromethane. Similarly also a second article of Moenius et al. (J. Labelled Cpd. Radiopharm.42, 29-41 (1999)) teaches that dichloromethane is the solvent in the reaction.

It appears that the compounds of formula (3) are very reactive, and thus also very unstable compounds. This is reflected by the fact that the yields of the reaction with rapamycine are very low and the compound (3) is charged in high molar extent. Methods how to monitor the reactivity and/or improve the stability of compounds of general formula (3), however, do not exist.

Thus, it would be useful to improve both processes of making compounds of formula (3) and, as well, processes of their application in chemical synthesis.

xample 6: 40-O-[2-((2,3-dimethylbut-2-yl)dimethylsilyloxy)ethyl]rapamycin

In a 100 mL flask, Rapamycin (6 g, 6.56 mmol) was dissolved in dimethoxyethane (4.2 ml) and toluene (24 ml) to give a white suspension and the temperature was raised to 70°C. After 20 min, N,N-diisopropylethylamine (4.56 ml, 27.6 mmol) and 2-((2,3-dimethylbutan-2- yl)dimethylsilyloxy)ethyl trifluoromethanesulfonate (8.83 g, 26.3 mmol) were added in 2 portions with a 2 hr interval at 70°C. The mixture was stirred overnight at room temperature, then diluted with EtOAc (40 ml) and washed with sat. NaHC0₃ (30 ml) and brine (30 ml). The organic layer was dried with Na₂S0₄, filtered and concentrated. The cmde product was chromatographed on a silica gel column (EtOAc/heptane 1/1 ; yield 4.47 g).

Example 7: 40-O-(2-hydroxyethyl)-rapamycin [everolimus]

In a 100 mL flask, 40-O-[2-((2,3-dimethylbut-2-yl)dimethylsilyloxy)ethyl]rapamycin (4.47 g, 4.06 mmol) was dissolved in methanol (20 ml) to give a colorless solution. At 0°C, IN aqueous hydrochloric acid (2.0 ml, 2.0 mmol) was added and the mixture was stirred for 90 min. The reaction was followed by TLC (ethyl acetate/n-heptane 3 :2) and HPLC. Then 20 ml of saturated aqueous NaHC03 were added, followed by 20 ml of brine and 80 ml of ethyl acetate. The phases were separated and the organic layer was washed with saturated aqueous NaCl until pH 6/7. The organic layer was dried by Na₂S0₄, filtered and concentrated to yield 3.3 g of the product.

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SYNTHESIS

https://www.google.co.in/patents/WO1994009010A1

Example 8: 40-O-(2-Hydroxy)ethyl-rapamycin

a) 40-O-[2-(t-Butyldimethylsilyl)oxy]ethyl-rapamycin

A solution of 9.14 g (10 mmol) of rapamycin and 4.70 mL (40 mmol) of 2,6-lutidine in 30 mL of toluene is warmed to 60°C and a solution of 6.17 g (20 mmol) of 2-(t-butyldimethylsilyl)oxyethyl triflate and 2.35 mL (20 mmol) of 2,6-lutidine in 20 mL of toluene is added. This mixture is stirred for 1.5h. Then two batches of a solution of 3.08 g (10 mmol) of triflate and 1.2 mL (10 mmol) of 2,6-lutidine in 10 mL of toluene are added in a 1.5h interval. After addition of the last batch, stirring is continued at 60°C for 2h and the resulting brown suspension is filtered. The filtrate is diluted with ethyl acetate and washed with aq. sodium bicarbonate and brine. The organic solution is dried over anhydrous sodium sulfate, filtered and concentrated. The residue is purified by column chromatography on silica gel (40:60 hexane-ethyl acetate) to afford 40-O-[2-(t-butyldimethylsilyl)oxy]ethyl-rapamycin as a white solid: ¹H NMR (CDCl₃) δ 0.06 (6H, s), 0.72 (1H, dd), 0.90 (9H, s), 1.65 (3H, s), 1.75 (3H, s), 3.02 (1H, m), 3.63 (3H, m), 3.72 (3H, m); MS (FAB) m/z 1094 ([M+Na]⁺), 1022 ([M-(OCH₃+H₂O)]⁺).

b) 40-O-(2-Hydroxy)ethyl-rapamycin

To a stirred, cooled (0°C) solution of 4.5 g (4.2 mmol) of 40-O-[2-(t-butyldimethylsilyl)oxy]ethyl-rapamycin in 20 mL of methanol is added 2 mL of IN HCl. This solution is stirred for 2h and neutralized with aq. sodium bicarbonate. The mixture is extracted with three portions of ethyl acetate. The organic solution is washed with aq.

sodium bicarbonate and brine, dried over anhydrous sodium sulfate, filtered and

concentrated. Purification by column chromatography on silica gel (ethyl acetate) gave the title compound as a white solid:¹H NMR (CDCl₃) δ 0.72 (1H, dd), 1.65 (3H, s), 1.75 (3H, s), 3.13 (5H, s and m), 3.52-3.91 (8H, m); MS (FAB) m/z 980 ([M+Na]⁺), 926 ([M-OCH₃]⁺), 908 ([M-(OCH₃+H₂O)]⁺), 890 ([M-(OCH₃+2H₂O)]⁺), 876 ([M-(2CH₃OH+OH)]⁺), 858 ([M-(OCH₃+CH₃OH+2H₂O)]⁺).

MBA (rel. IC50) 2.2

IL-6 dep. prol. (rel. IC50) 2.8

MLR (rel. IC50) 3.4

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synthesis

Everolimus (Everolimus) was synthesized by the Sirolimus (sirolimus, also known as rapamycin Rapamycin) ether from. Sirolimus is from the soil bacterium Streptomyces hygroscopicus isolated metabolites. Activation end sirolimus (triflate, Tf) the other end of the protection (t-butyldimethylsilyl, TBS) of ethylene glycol 1 reaction of 2 , because the hydroxyl group 42 hydroxyl site over the 31-bit resistance is small, so the reaction only occurs in 42. Compound 2under acidic conditions TBS protection is removed everolimus.